In 2013, the CDC designated carbapenem-resistant Enterobacteriaceae (CRE) an Urgent Threat, and in 2017, the WHO designated it a Priority 1 ?critical superbug?. As few therapies remain to treat CRE, the risk of ?pan- resistant? CRE untreatable by any currently available antibiotic also exists. Entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. Development of such a ?first-in-class? agent also offers the potential for much-needed orally-administered CRE therapeutic, thus providing a new step-down therapy to reduce hospital stay and growing healthcare costs. Our proposal aims to develop an entirely novel therapeutic to treat life-threatening bacterial infections due to multidrug resistant (MDR) Enterobacteriaceae, including CRE. Using an innovative overexpression-based co-culture screen in Escherichia coli (Ec), we identified four structurally distinct series of small molecule inhibitors targeting MsbA, an essential and broadly conserved Gram-negative (GN) ABC transporter responsible for lipopolysaccharide (LPS) biogenesis and construction of the Gram-negative outer membrane (OM). Building upon a solid foundation of preliminary data, our Aims are: Aim (A) 1. Expanded MOA studies. Aim 1 studies will expand upon our preliminary MOA and microbiological characterization of the MsbA inhibitor (MsbAi) hits to include other GN bacteria, particularly Klebsiella pneumoniae (Kp). Specifically, we will demonstrate Kp MsbA target inhibition in (1) an in vitro biochemical assay and (2) a whole-cell context by determining MICs of our MsbAis against a Kp ?tolC strain and subsequently selecting for MsbAisR mutants in this Kp background. Aim 2. Demonstrate chemical tractability of MsbAis. The goal of Aim 2 is to perform Hit to Lead (H2L) medicinal chemistry structure activity relationship (SAR) studies for each MsbAi and demonstrate >1 series is chemically tractable and that quantifiable pre-lead optimization criteria can be established, including i) Ec and Kp MsbA in vitro potency (IC50 < 0.25 g/ml), ii) Ec and Kp WT activity (<16 g/ml), and iii) > 90% HepG2 cell viability at 50X MIC against Ec?tolC. MsbAis achieving these milestones serve as a justifiable starting point for a future Lead Optimization leveraging the resulting SAR gained in Aim 2 and SBDD information from Aim 3. Aim 3. Obtain Ec and Kp MsbAi-MsbA co-crystal structures in collaboration with SSGCID. The goal of Aim 3 activities is to initiate a collaboration between Prokaryotics, Genentech, and the Seattle Structural Genomics Center for Infectious Disease (SSGCID) led by Dr. Peter Myler to optimize conditions suitable for obtaining high resolution x-ray crystal structures of Kp MsbA in the apo form as well as Ec and/or Kp MsbA proteins in complex with MsbAis.